Notes

From There and also Rear.
In addition, we describe fluorescent staining specific for O2- detection and mitochondrial membrane potential, as well as a simple clonogenic assay based on the ability of cells to grow on a carbon source that requires a functional mitochondrial metabolism.This chapter presents a protocol for assessing the effects dietary seafood consumption on depressive symptoms. We designed a cross-sectional study of 206 participants recruited in two Torres Strait Island communities. Depressive symptoms were assessed using the adapted Patient Health Questionnaire-9 (aPHQ-9), diet was analyzed with a structured questionnaire, omega-3 and omega-6 fatty acid concentrations were measured via a capillary dried blood spot system, and plasma levels of triglycerides and cholesterol were measured by gas-phase chromatography. Finally, we tested the relationship between seafood consumption, blood lipid concentrations, and depression scores using independent samples t-tests and a logistic and quantile regression model.This chapter describes the application of two-dimensional gel electrophoresis (2DGE) combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in the analysis of rat eye lens proteins. The main purpose was to identify proteins that may serve as potential biomarkers in age-related cataract formation. This includes the family of proteins known as the crystallins. Structural proteins and enzymes involved antioxidant activities. find more In addition, we also analyzed lenses from other species to illustrate the potential of using this technique in clinical and preclinical biomarker studies.Nicotinamide adenine dinucleotide (NAD+) and its related metabolites (NADome) are important endogenous analytes that are thought to play important roles in cellular metabolism, inflammation, oxidative stress, cancer, neurodegeneration, and aging in mammals. However, these analytes are unstable during the collection of biological fluids, which is a major limiting factor for their quantitation. Herein, we describe a highly robust and quantitative method using liquid chromatography coupled to tandem mass spectrometry to quantify the NADome in whole blood, plasma, mononuclear cells, platelets, cerebrospinal fluid (CSF), and urine. This methodology represents a "gold standard" of measure for understanding biological pathways and developing targeted pharmacological interventions to modulate NAD+ biosynthesis and NAD-dependent mediators in health and disease.In the nematode Caenorhabditis elegans, the mammalian tumor suppressor p53 ortholog CEP-1 (C. elegans p53-like protein) is associated not only with the stress response, germline apoptosis, and meiotic chromosome segregation but also with longevity through the modification of energy metabolism during aging. The mitochondrial respiration-related gene sco-1 in C. elegans is orthologous to the human SCO1 gene and a target of p53/CEP-1. Using quantitative real-time polymerase chain reaction (PCR) analysis, we recently found that the expression levels of sco-1 gene were increased in wild-type C. elegans in an aging-related manner and decreased in long-lived cep-1 mutants. Here, we describe the relative quantitative strategy using a commercial real-time PCR system to detect more accurately differences in the levels of expressed genes between long-lived and wild-type C. elegans strains. To estimate the expression levels of target genes compared with wild-type using relative quantification, we used the expression levels of an endogenous control gene, such as a housekeeping gene. In addition, it is critical to normalize differences in the expression levels of the common housekeeping gene among the strains analyzed for an accurate comparison of the quantitative expression levels of target genes.Acquisition of anoikis resistance is a prerequisite for cancer metastasis and invasion, which are major causes of death from cancer. The molecular mechanisms underlying antianoikis properties in cancer cells are still largely unclear. Here, we describe a protocol for preparation of anoikis-resistant cultured nonmetastatic MCF-7 and metastatic MDA-MB-231 cell lines. The anoikis-resistant cultures were prepared by plating cells in the poly-2-hydroxyethyl methacrylate coated plates and cultured for 24 h. The viability of cells in the cultures was determined using trypan blue staining and annexin V cell death assay, while protein profiles associated with anoikis-resistance in both cells and conditioned media were analyzed by proteomics.Electrochemical biosensors provide rapid, selective, and sensitive diagnostic platforms for detecting and monitoring biochemical processes in living systems in vivo and in vitro, and have been widely applied in various fields of biology and medicine. Sorafenib is a multi-kinase inhibitor used as a standard therapy for advanced hepatocellular carcinoma (HCC). However, the molecular basis for sorafenib resistance in HCC remains elusive. Recently, we developed new protocols for an electrochemical biosensor and applied these to monitor the levels of superoxide and nitric oxide produced in HCC cells, in the presence or absence of sorafenib. We also employed electrochemical biosensor to determine the release of profiles of superoxide and nitric oxide in sorafenib-treated HCC cells under the influence of fibroblast growth factor 19 expression levels. Here we present protocols to highlight the utility of electrochemical strategies in drug and gene studies.With the development of new materials and technologies, it is possible to access gene function and drug metabolism using a three-dimensional (3D) cell culture system, which is more suitable for mimicking the in vivo microenvironment of cultured tumor cells ex vivo. SeedEZ is a novel and versatile tool that allows culturing of different types of cells with user convenience and in a desired sequence. This system provides a bridge between traditional 2D culture and animal experiments. Here, we provide two examples demonstrating how to evaluate cancer cell growth by the SeedEZ 3D scaffold and cancer cell invasion by the SeedEZ 3D ring, in order to promote understanding of the necessity of this novel cell culture system.
Homepage: https://www.selleckchem.com/products/AZD1480.html